Antibodies are protein molecules which specifically bind antigens that initiate the formation of the antibodies. Antibodies are produced by B-lymphocytes and plasma cells, and are present in blood plasma (as secretory antibodies or cell bound), lymphatic fluids, cerebrospinal fluid, mucus and other extracellular secretions (e.g., saliva), and in some instances, urine. Assays for antibodies have been widely applied to the examination and diagnosis of many infections, and autoimmune and allergic conditions.
There are currently several known immunoglobulin classes, IgG, IgM, IgA, IgE and IgD. These immunoglobulins are involved in different types of immune responses, and their involvements are dependent, for example, on antigen structures, type of organisms which bear these antigens, time after immunization, and presence of other antigenic molecules in the microorganisms. Antibody classes important for serological testing, primarily secretory immunoglobulins, contain two or more identical antigen-combining sites. In particular, there are two antigen combining sites on IgG and IgE, four on IgA, and up to 10 on IgM. This means that at least two antigen molecules or specific antigenic (epitopic) substances can bind simultaneously to one antibody molecule.
It should be noted that affinity of antibodies to specific antigens may not be constant. Beside the spectrum of polyclonal antibodies which can differ in affinity at varying stages of an immune response depending on the particular pathogen/immunogen, affinity of antibodies can change with time, from low to high affinity, for instance, during the antibody maturation process. Moreover, several antibody classes can simultaneously recognize the same antigen. In the case of bacterial, viral, protozoan, parasitic, or fungal infection, the IgM class can appear at the early stages of an immune response, usually as antibody with a low affinity constant, while the IgG class can appear later in the immune response. For serological testing of many infections, the ability to detect antibodies at the earliest stages of seroconversion can be very important in determining a treatment. Because the IgM class antibodies often appear during the early stages of infection, their presence is often considered as a serological indication of early stage of infection.
Antibodies can be detected using a variety of immunoassay protocols. Among conventional methods, the most widely used works on principles of solid phase immunoassay, using immobilized antigens and secondary class-specific anti-immunoglobulins conjugated with detector labels, such as enzyme, fluorophores, chemiluminescent, metal sols, dyed polymeric particles, or labeled proteins which bind to immunoglobulins (protein A and G). A disadvantage of such a method is that a falsely positive result can be obtained due to non-specific and cross-reactive reactions related to simple adsorption of non-specific antibodies on coated solid phase, or the presence of unwanted antigenic substances in antigens used for adsorption on solid phase. This can happen even in case of using relatively pure antigens obtained by methods of recombinant technology. Moreover, the presence of, for instance, autoantibodies, rheumatoid factor, or polyspecific/polyreactive antibodies, can lead to false results. Even in case of using very specific small peptide antigens, false positives can arise as result of contaminations in the reagents used for immobilization on solid phase.
In order to reduce the occurrence of false positives, steps such as selection of sample dilution, incubation time, temperature, and diluents used, have been taken to reduce potential cross-reactive reactions. However, such steps can require the application of many serological tests and can reduce detection sensitivity. For example, in a solid phase immunoassay using solid phase immobilized antigens, the presence of conjugates antigen-specific antibodies with detector label can compete with antibodies in a sample to be detected to bind with the immobilized antigens. As a result, although this competitive test is relatively simple and may be less susceptible to non-specific reactions, its sensitivity for detection can be relatively low.
In another immunoassay for specific antibody detection based on competitive binding, a specific antibody may be immobilized in a solid phase. However, during incubation, labeled antigens in the sample and the antibody to be detected in the sample can compete to bind with the immobilized antibody. As with the previous example, sensitivity of detection may be affected due to competitive binding, and as a result, may require a significant amount of pure anti-antigen antibodies for use.
In another immunoassay, the solid phase may be coated with a class specific immunoglobulins. Labeled antigens may be used in the sample of the antibody to be detected to form an immune complex with the class specific immunoglobulins in the solid phase. One disadvantage of this immunoassay is the limited binding capacity of the class specific immunoglobulins in the solid phase, which can limit the sensitivity of specific antibody detection.
A further variation of solid phase immunoassay involves the use of solid phase coated antigens to capture specific antibody and subsequent detection of captured/bound antibody with a labeled antigen. However, a prozoning phenomenon may be a problem for this approach. In particular, the density of antigen on solid phase should be carefully optimized to protect the antibody from binding with the solid phase antigen through all combining sites (e.g., IgG class) on the antibody, which reduce number of sites available on the antibody to bind with labeled antigen. A second incubation with labeled antigen is described in U.S. Pat. No. 6,121,006 as a way for increasing sensitivity of this type of assay.
U.S. Pat. No. 6,030,770 discloses a method for antibody detection using labeled antigen and a solid phase capture system. In this method, the solid phase comprises immobilized antigens and anti-immunoglobulin antibodies introduced into the capture system through an additional bridge. The bridge, as disclosed, includes analyte-specific antibodies capable of recognizing ligand-labeled anti-species class specific antibodies.
U.S. Pat. No. 4,778,751 discloses a method for detection of circulating antibodies, which method is based on the use of a ligand-labeled polymeric matrix. In this method, the matrix is conjugated with multiple antigen molecules, a ligand-binding partner immobilized on solid phase, and anti-immunoglobulin detector reagent.
In U.S. Pat. No. 4,945,042, a three reagents antibody detection system is disclosed. The detection system, as disclosed, comprises two components of a specific binding pair. One component is included in the capture system as a conjugate of StrAv with thermo polymerized BSA of anti-immunoglobulin antibodies, while the second component is conjugated to the antigen. A third reagent is labeled and is a conjugate of the antigen.
U.S. Pat. No. 5,236,849 discloses a detection method utilizing an antigen containing simultaneously two different labels belonging to two affinity pairs, each with its counterparts distributed between separate solid phases. This approach permits for significant reduction of non-specific reaction by using immune-complex transfer (dissociation-recapture) for detection in a second container.
Lyphophilic bridges, such as liposomes or some other reversible bridge, are disclosed in U.S. Pat. Nos. 5,312,730 and 5,705,338 as a way to dissociate an immune complex from a first solid phase to permit transfer into a second solid phase for detection.
Small peptide antigens, containing specific epitopes, mono or limited amount, can be powerful instrument for development of highly specific serological test. The roles of these peptides, whether obtained by chemical synthesis or recombinant technology, have become increasingly important. However, affinity of antibodies against these small epitopes is sometimes lower than the affinity to a sequence in whole protein. Moreover, affinity of antibodies to selected peptide antigens can vary due to the presence of various strains of pathogens and their genetic variability. Accordingly, it is desirable to provide simple methods, which can permit the detection of antibodies that may have low affinity to selected epitopes.